1. Field of the Invention
The present invention relates to methods for the generation of lambda (xcex) or P1 bacteriophage vectors useful in targeted mutagenesis of eukaryotic cells and the expression of genes and proteins, methods for the identification of a xcex or P1 bacteriophage vector having a desired nucleic acid from an assortment or library of bacteriophage each having a different nucleic acid insert and the use of such vectors in gene targeting and the expression of genes and protein.
2. Description of the Related Art
The present invention provides a method for the construction of a xcex or P1 bacteriophage vector using bacteriophage/plasmid recombination and selection for double-crossover bacteriophage recombinants.
Vectors have traditionally been generated through restriction enzyme digestion of the vector and religation with the desired target nucleic acid. In general, two types of problems are encountered in the construction of vectors by this method. First, plasmid vectors may be undesirable because specific eukaryotic genomic regions can undergo rearrangements in plasmid vectors. Therefore genomic regions may be difficult to clone either on their own or in combination with eukaryotic selectable marker genes, such as neo or tk. Secondly, larger sizes of target DNA sequences are desirable. However, the larger the DNA sequence, the more restriction enzyme sites present in the DNA. For cloning purposes, suitable restrictions sites are low frequency sites located on either side of the target nucleic acid sequence. Therefore, because of the large number of restriction enzyme sites in large genomic fragments, the use of such fragments in vectors means that there is often very few or no suitable restriction sites for inserting foreign DNA fragments, such as positive selectable marker genes or small mutations.
Accordingly, it would be desirable to develop a method for the generation of vectors capable of accepting large genomic fragments without rearrangement and without the need for suitable restriction enzyme sites.
Bacteriophage/plasmid recombination has been used to screen and isolate targeted xcex phage from genomic libraries (15, 17). For example, a xcex genomic library (bearing amber mutations) was passaged over a rec+ bacterial strain bearing a small supF (amber suppressing) recombination plasmid having sequence homologous to the desired gene. Homology in the recombination plasmid, usually derived from a cDNA sequence, directed the plasmid to integrate into the phage by a single crossover, thereby generating supF bearing phage recombinants capable of growing on a suppressor free (sup0) host. Depending on homology length, the recombination plasmid can integrate at a frequency of xcx9c10xe2x88x922. One of the difficulties with this method of bacteriophage/plasmid recombination is that it generated single cross-over recombinants. Single cross-over recombinants are generally considered undesirable because of the presence of plasmid sequences and the partial duplication of the target nucleic acid.
Accordingly, it would be beneficial to develop a method for the identification of a recombinant bacteriophage from a library through plasmid/phage recombination which method resulted in the isolation of the original bacteriophage without the insertion of the plasmid nucleic acid sequences.
Eukaryotic gene targeting involves the selection for homologous recombination events between DNA sequences residing in the genome of a eukaryotic cell or organism and newly introduced DNA sequences. This provides a means for systematically altering the genome of a eukaryotic cell or organism. For mammalian systems, laboratories have reported the insertion of exogenous DNA sequences into specific sites within the mammalian genome by way of homologous recombination. For example, targeted mutagenesis allows specific mutations to be engineered into the mouse germline via homologous recombination of exogenously-altered DNA in embryonic stem (ES) cells (1, 2). Using this technology, the function of any cloned gene may be examined by its disruption in mice. Thus, gene targeting is a critical experiment in molecular medicine, and is used, for example, to mimic human mutations in the mouse for the generation of experimental therapeutic models (3).
The original and still the most prevalent gene targeting approach ,xe2x80x9cthe knockoutxe2x80x9d, uses a replacement plasmid vector to direct a positive selectable marker (i.e. neomycin resistance gene) into a specific chromosomal location via either double-reciprocal exchange or gene conversion (4). Positive-negative selection vectors have been used for gene targeting (26). Many sophisticated variations on this original technique have become available, including the generation of point mutations, deletions and translocations and gene substitutions (5-9). Further, the application of cre recombinase from bacteriophage P1 allows additional genomic alterations at loxP target sequences following gene targeting so that mutations can be made tissue- or development-specific (10).
Although targeted mutagenesis provides a powerful tool for the analysis of gene function, it is a complex and time-consuming procedure. While methods of improving the efficiency of generating targeted ES cell lines (11) and mutant mice (12) have become available, little has been done to streamline the construction of the targeting vector. Currently the rate determining step in any gene targeting experiment is the construction of the targeting vector.
Accordingly, there is a need to develop a method to generate targeting vectors which does not require as much cumbersome restriction enzyme methodology and yet would yield targeting vectors which are efficient in inserting larger fragments of the modified nucleic acid into the desired site in the eukaryotic cell.
Further advantages of the present invention will become apparent from the following description of the invention with reference to the attached drawings.
This invention describes how double-crossover bacteriophage generated by bacteriophage/plasmid recombination can be selected through the use of double-crossover selectable markers present on the plasmid vector. The present invention is directed to the generation of xcex or P1 bacteriophage vectors using this method. The present invention is also directed to a method for screening a xcex or P1 bacteriophage library for the identification of a recombinant bacteriophage having the desired target sequence and to the generation of bacteriophage targeting vectors.
One aspect of this invention is directed to a method for generating recombinant xcex or P1 bacteriophage vectors, which method comprises
(a) providing a xcex or P1 bacteriophage nucleic acid sequence comprising a first target nucleic acid sequence;
(b) providing a plasmid comprising a nucleic acid sequence encoding a second modified target nucleic acid sequence, and a double-crossover selectable marker gene wherein the second modified target nucleic acid sequence is substantially homologous over a portion of its length to the first target nucleic acid sequence;
(c) contacting the bacteriophage and the plasmid under conditions such that homologous recombination between the first target nucleic acid sequence and the second target nucleic acid sequence occurs;
(d) selecting for double-crossover recombinant bacteriophage by placing the bacteriophage from step (c) under conditions such that bacteriophage having the double-crossover selectable marker are unable to replicate and isolating the double-crossover recombinant bacteriophage. The double-crossover selectable marker gene may be gam where the xcex recombinant bacteriophage is grown in a P2 lysogenic bacterial cell. The double-crossover selectable marker may be any large nucleic acid sequence where the xcex or P1 recombinant bacteriophage is placed under a size restriction, such as a requirement to be packaged in a viral coat or particle.
In this method, the plasmid may further comprise a prokaryotic positive selectable marker inserted into the target nucleic acid sequence. The prokaryotic positive selectable marker is preferably supF or supE genes where the bacteriophage has amber mutations in essential genes. The plasmid may further comprise a eukaryotic positive selectable marker inserted into the target nucleic acid sequence. Preferably, the eukaryotic positive selectable marker gene may be the Neo, Hyg, hisD, Gpt, Ble,or Hprt genes. The bacteriophage may further comprise a eukaryotic negative selectable marker. Preferably, the eukaryotic negative selectable marker is the tk1 or tk2 genes.
Another aspect of this invention is a recombinant xcex or P1 bacteriophage nucleic acid comprising a nucleic acid sequence encoding a positive eukaryotic selectable marker located within a target nucleic acid sequence and a positive prokaryotic selectable marker. Preferably the bacteriophage further comprises a nucleic acid sequence encoding a negative eukaryotic selectable marker positioned 5xe2x80x2 or 3xe2x80x2 to the target nucleic acid sequence. Also contemplated are bacteriophage particles comprising the recombinant bacteriophage nucleic acid. Also contemplated is a bacteriophage wherein the target nucleic acid sequence is further modified by insertions, deletions or substitutions.
In another aspect of this invention the recombinant bacteriophage nucleic acid is isolated from the bacteriophage particle and restriction enzyme digested to remove the bacteriophage arm nucleic acid.
Another aspect of this invention is a method for insertion of a modified target nucleic acid sequence into a eukaryotic cell genome through homologous recombination comprising providing a recombinant bacteriophage nucleic acid comprising a nucleic acid sequence encoding a positive eukaryotic selectable marker gene located within a modified target nucleic acid sequence; and contacting a eukaryotic cell with the bacteriophage nucleic acid under conditions whereby the modified target nucleic acid sequence in the bacteriophage undergoes homologous recombination with the target nucleic acid sequence in the eukaryotic cell. Preferably the bacteriophage further comprises a nucleic acid sequence encoding a negative eukaryotic selectable marker gene positioned 5xe2x80x2 or 3xe2x80x2 to the target nucleic acid. Preferably, the recombinant bacteriophage nucleic acid is packaged in a virion particle prior to contacting the eukaryotic cell.
Another aspect of this invention is a method for selection of a bacteriophage having a desired target nucleic acid sequence from an assortment of bacteriophage wherein each of the bacteriophage comprise a different nucleic acid insert comprising the following steps:
providing a plasmid, which plasmid comprises a portion of the desired target nucleic acid sequence, a positive selectable marker gene and a double-crossover selectable marker,
providing an assortment of bacteriophage comprising different nucleic acid inserts;
contacting the assortment of bacteriophage with the plasmid under conditions such that homologous recombination between the target nucleic acid sequence on the plasmid and the desired target nucleic acid sequence on the bacteriophage can occur;
growing the bacteriophage in bacterial cells under conditions wherein those bacteriophage which have recombined with the plasmid are able to replicate; growing the bacteriophage in bacterial cells under conditions wherein those bacteriophage lacking the double cross-over selectable marker are able to replicate; and identifying those bacteriophage as comprising the desired target nucleic acid.